Epidemic keratoconjunctivitis is caused by human adenovirus species D (HAdV-D) types 8, 19, and 37, and more recently also 53 and 54. The clinical manifestations of this infection include severe pseudomembranous conjunctivitis and epithelial keratitis, followed by multifocal subepithelial (stromal) corneal infiltrates that cause photophobia and reduced vision and may persist for months to years. Previous studies focused on cellular and nuclear signaling events that lead to expression of proinflammatory mediators such as IL-8 and MCP- 1 in the corneal stroma. However, it is expected that signaling events also control entry of the virus and its intracellular route. Lipid rafts, including caveolin containing endosomes that bud off from the cell membrane during viral internalization, and signaling molecules associated with them, have proven to be viable targets for therapy against viral infections, including human immunodeficiency virus. The role(s) of lipid rafts in adenovirus infections has not been extensively studied, and has never previously examined in ocular adenovirus infection. Therefore, the objective of this proposal is to elucidate how lipid rafts and caveosomes in corneal cells influence entry and trafficking of cornea tropic HAdV-D. As specific aims, three hypotheses will be tested: 1) lipid raft induced cell signaling is necessary for HAdV-D entry into corneal cells, 2) caveosomes play a role in intracellular trafficking of HAdV-D, and 3) lipid rafts and caveolin are necessary for HAdV-D infection in the mouse model of adenovirus keratitis. These studies will utilize human primary corneal cells, i.e., those that are actually infected by the virus during ocular infection, and the mouse model of adenovirus keratitis developed previously, with a special focus on the caveolin knockout mouse. Individual experiments will include use of transfected siRNA and dominant negative kinases against caveolin, dynamin, and protein kinases, confocal microscopy and live cell imaging to visualize viral internalization and intracellular trafficking, and polymerase chain reaction to confirm viral entry and gene expression. The current proposal addresses a major programmatic goal in the National Plan for Eye and Vision Research, to "investigate corneal infectious and inflammatory processes and immunological responses to develop treatments to reduce keratitis and prevent blindness". As adenoviruses represent the most common cause of eye infections, this proposal will address a major public health concern. PUBLIC HEALTH RELEVANCE: Ocular surface infection by adenoviruses represent the most common type of eye infection, and affected patients suffer considerably from associated blurred vision and discomfort. However, there is at present no specific therapy against adenovirus infection. This proposal directly addresses the means by which adenoviruses enter cells of the cornea, specifically the role of a specialized aspect of the cell membrane, termed lipid raft, and will provide sufficient understanding to design future therapies against adenovirus infection.